Sarcopenia, which diminishes lifespan and healthspan in the elderly, is commonly exacerbated by viral pneumonia, including influenza and COVID-19. In a study of influenza A pneumonia in mice, young mice fully recovered from sarcopenia, while older mice did not. We identified a population of tissue-resident skeletal muscle macrophages that form a spatial niche with satellite cells and myofibers in young mice but are lost with age. Mice with a gain-of-function mutation in the Mertk receptor maintained this macrophage-myofiber interaction during aging and fully recovered from influenza-induced sarcopenia. In contrast, deletion of Mertk in macrophages or loss of Cx3cr1 disrupted this niche, preventing muscle regeneration. Heterochronic parabiosis did not restore the niche in old mice. These findings suggest that age-related loss of Mertk in muscle tissue-resident macrophages disrupts the cellular signaling necessary for muscle regeneration after viral pneumonia, offering a potential target to mitigate sarcopenia in aging.

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here — using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures — we show that hypercapnia limits β-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize β-catenin signaling, thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.

ABSTRACTPersistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, we show that hypercapnia limits β-catenin signaling in alveolar type 2 (AT2) cells, leading to reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRa+-fibroblasts from those maintaining AT2 progenitor activity and towards those that antagonize β-catenin signaling and limit progenitor function. Activation of β-catenin signaling in AT2 cells, rescues the inhibition AT2 proliferation induced by hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality.

Epithelial polyploidization after injury is a conserved phenomenon recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces alveolar type 2 cell (AT2) hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 transdifferentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. Because AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 selfrenewal and regenerative potential upon reinjury, which may benefit from targeting the integrated stress response.

Muscle dysfunction often occurs in patients with chronic obstructive pulmonary diseases (COPD) and affects ventilatory and non-ventilatory skeletal muscles. We have previously reported that hypercapnia (elevated CO2 levels) causes muscle atrophy through the activation of the AMPKα2-FoxO3a-MuRF1 pathway. In the present study, we investigated the effect of normoxic hypercapnia on skeletal muscle regeneration. We found that mouse C2C12 myoblasts exposed to elevated CO2 levels had decreased fusion index compared to myoblasts exposed to normal CO2. Metabolic analyses of C2C12 myoblasts exposed to high CO2 showed increased oxidative phosphorylation due to increased fatty acid oxidation. We utilized the cardiotoxin-induced muscle injury model in mice exposed to normoxia and 10% CO2 for 21 days and observed that muscle regeneration was delayed. High CO2-delayed differentiation in both mouse C2C12 myoblasts and skeletal muscle after injury and was restored to control levels when cells or mice were treated with a carnitine palmitoyltransfearse-1 (CPT1) inhibitor. Taken together, our data suggest that hypercapnia leads to changes in the metabolic activity of skeletal muscle cells, which results in impaired muscle regeneration and recovery after injury.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-kB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-kB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Carbon dioxide (CO2) is produced in eukaryotic cells primarily during aerobic respiration, resulting in higher CO2 levels in mammalian tissues than those in the atmosphere. CO2 like other gaseous molecules such as oxygen and nitric oxide, is sensed by cells and contributes to cellular and organismal physiology. In humans, elevation of CO2 levels in tissues and the bloodstream (hypercapnia) occurs during impaired alveolar gas exchange in patients with severe acute and chronic lung diseases. Advances in understanding of the biology of high CO2 effects reveal that the changes in CO2 levels are sensed in cells resulting in specific tissue responses. There is accumulating evidence on the transcriptional response to elevated CO2 levels that alters gene expression and activates signaling pathways with consequences for cellular and tissue functions. The nature of hypercapnia-responsive transcriptional regulation is an emerging area of research, as the responses to hypercapnia in different cell types, tissues, and species are not fully understood. Here, we review the current understanding of hypercapnia effects on gene transcription and consequent cellular and tissue functions.

Carbon dioxide (CO2) is sensed by cells and can trigger signals to modify gene expression in different tissues leading to changes in organismal functions. Despite accumulating evidence that several pathways in various organisms are responsive to CO2 elevation (hypercapnia), it has yet to be elucidated how hypercapnia activates genes and signaling pathways, or whether they interact, are integrated, or are conserved across species. Here, we performed a large-scale transcriptomic study to explore the interaction/integration/conservation of hypercapnia-induced genomic responses in mammals (mice and humans) as well as invertebrates (Caenorhabditis elegans and Drosophila melanogaster). We found that hypercapnia activated genes that regulate Wnt signaling in mouse lungs and skeletal muscles in vivo and in several cell lines of different tissue origin. Hypercapnia-responsive Wnt pathway homologues were similarly observed in secondary analysis of available transcriptomic datasets of hypercapnia in a human bronchial cell line, flies and nematodes. Our data suggest the evolutionarily conserved role of high CO2 in regulating Wnt pathway genes.

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1+/- and atrogin-1-/- mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome.

Cardiac glycosides (CGs) are used primarily for cardiac failure and have been reported to have other effects, including inhibition of viral replication. Here we set out to study mechanisms by which CGs as inhibitors of the Na-K-ATPase decrease influenza A virus (IAV) replication in the lungs. We found that CGs inhibit influenza virus replication in alveolar epithelial cells by decreasing intracellular potassium, which in turn inhibits protein translation, independently of viral entry, mRNA transcription, and protein degradation. These effects were independent of the Src signaling pathway and intracellular calcium concentration changes. We found that short-term treatment with ouabain prevented IAV replication without cytotoxicity. Rodents express a Na-K-ATPase-α1 resistant to CGs. Thus we utilized Na-K-ATPase-α1-sensitive mice, infected them with high doses of influenza virus, and observed a modest survival benefit when treated with ouabain. In summary, we provide evidence that the inhibition of the Na-K-ATPase by CGs decreases influenza A viral replication by modulating the cell protein translational machinery and results in a modest survival benefit in mice.

The elevation of carbon dioxide (CO2) in tissues and the bloodstream ( hypercapnia) occurs in patients with severe lung diseases, including chronic obstructive pulmonary disease (COPD). Whereas hypercapnia has been recognized as a marker of COPD severity, a role for hypercapnia in disease pathogenesis remains unclear. We provide evidence that CO2 acts as a signaling molecule in mouse and human airway smooth muscle cells. High CO2 activated calcium-calpain signaling and consequent smooth muscle cell contraction in mouse airway smooth muscle cells. The signaling was mediated by caspase-7-induced down-regulation of the microRNA-133a(miR-133a) and consequent up-regulation of Ras homolog family member A and myosin light-chain phosphorylation. Exposure of wildtype, but not caspase-7-null, mice to hypercapnia increased airway contraction and resistance. Deletion of the Caspase-7 gene prevented hypercapnia-induced airway contractility, which was restored by lentiviral transfection of a miR-133a antagonist. In a cohort of patients with severe COPD, hypercapnic patients had higher airway resistance, which improved after correction of hypercapnia. Our data suggest a specific molecular mechanism by which the development of hypercapnia may drive COPD pathogenesis and progression.

Patients with chronic obstructive pulmonary disease may develop hypercapnia. Many of these patients have muscle dysfunction which increases morbidity and impairs their quality of life. We have previously reported that mice with normoxic-hypercapnia (pCO2:77 mmHg) have skeletal muscle atrophy with reduced body weight and muscle cross-sectional area. In the present study, we examined the effect of hypercapnia on skeletal muscle repair and, in particular, in skeletal muscle differentiation. In mice exposed for 21 days to 10% CO2, we found that muscle recovery after cardiotoxin injury was delayed as determined by increased number of centralized nuclei and expression of embryonic myosin heavy chain. In vitro experiments revealed that mouse C2C12 myoblasts exposed to high CO2 displayed a significant impairment in myotube formation compared to myoblasts exposed to normocapnia, independently of pH. Metabolome analysis of myoblasts exposed to high CO2 revealed increased oxidative phosphorylation due to increased fatty acid oxidation. Mice and myoblasts phenotype was restored to normoxic levels with the treatment of the selective fatty acid import inhibitor etomoxir. However, the impaired differentiation into myotubes persisted even when the myoblasts were switched from high CO2 to normal CO2 conditions suggesting a potential hypercapnia-induced epigenetic imprinting. Taken together, our data suggest that hypercapnia leads to changes in the metabolic activity of skeletal muscle cells, which results in impaired myotube formation and recovery after injury. Supported by NIH HL-085534, HL-48129 and AG-049665

Significance Exposure to hypoxia requires adaptive mechanisms for survival. During acute hypoxia, Na,K-ATPase endocytosis in alveolar epithelial cells occurs via protein kinase C zeta (PKCζ) phosphorylation of α 1 -Na,K-ATPase independently of the hypoxia-inducible factor (HIF). However, exaggerated Na,K-ATPase down-regulation leads to cell death. Here we report that during prolonged hypoxia plasma membrane Na,K-ATPase levels were maintained at ∼50% of normoxic values due to HIF-mediated up-regulation of HOIL-1L, which targets PKCζ for degradation. Silencing HOIL-1L in the lung epithelium prevented PKCζ degradation, causing Na,K-ATPase downregulation. Accordingly, HIF regulation of HOIL-1L targets the phosphorylated PKCζ for degradation and serves as an hypoxia-adaptive mechanism to stabilize the Na,K-ATPase, avoiding significant lung injury.

The ability of organisms to adapt to hypoxia is critical for survival. Cells down regulate energy consuming enzymes such as Na,K‐ATPase, in order to decrease O2 (oxygen) consumption. The Na,K‐ATPase activity is crucial for the alveolar epithelium function and cell homeostasis. In alveolar epithelial type 2 (ATII) cells exposed to short term hypoxia, Na,K‐ATPase abundance at the plasma membrane is tightly regulated by protein kinase C zeta (PKCζ) through phosphorylation of its α1 catalytic subunit at Ser‐18 which triggers its endocytosis decreasing the enzyme's activity. We have previously described that during prolonged hypoxia, HIF promotes transcription of HOIL‐1L, which acts as the E3‐ubiquitin ligase for PKCζ targeting it for proteasomal degradation. In the present study, we focused on elucidating whether PKCζ degradation by HOIL‐1L prevents further Na,K‐ATPase down‐regulation as a mechanism of adaptation to hypoxia. C57BL/6 WT mice or SPC‐Cre‐HOIL‐1Lfl/fl mice, which have a lung epithelial specific deletion of HOIL‐1L, were exposed to room‐air or hypoxia (7% O2) for up to 14 days. Protein expression in ATII cells was analyzed by Western blot. We found that hypoxia‐induced PKCζ degradation was prevented in SPC‐Cre‐HOIL‐1Lfl/fl mice. Moreover, the absence of HOIL‐1L failed to prevent plasma membrane Na,K‐ATPase depletion as compared to hypoxia‐exposed WT mice. The alveolo‐capillary barrier permeability to small solutes, measured by FITC‐labeled dextran (4KDa), cell count and protein content in broncho‐alveolar lavage fluid were increased in SPC‐Cre‐HOIL‐1Lfl/fl mice. Also, histological analysis showed an increased thickening in the alveolar septa and apoptosis in SPC‐Cre‐HOIL‐1Lfl/fl mice after 7 days of hypoxia, suggesting that HOIL‐1L is crucial for adaptation to hypoxia. Interestingly, the increased epithelial barrier damage observed due to the lack of HOIL‐1L was independent of the inflammatory response. In AEC exposed to 1.5% O2 in vitro, the silencing of HOIL‐1L led to Na,K‐ATPase downregulation and cell death as compared to cells transfected with control siRNA, and was rescued by transfection with PKCζ phosphorylation resistant S18A‐α1 Na,K‐ATPase. To further explore the mechanism leading to PKCζ degradation, PKCζ interaction with HOIL‐1L was studied using gain and loss of function mutations in the kinase activation loop. Our results suggest that hypoxia‐induced phosphorylation of PKCζ T410 is required for the hypoxia‐induced translocation to the plasma membrane and interaction with HOIL‐1L, which triggers PKCζ ubiquitination and degradation. Collectively, these data provide evidence for an adaptive mechanism to hypoxia where the coordinated action of HOIL‐1L and PKCζ regulates Na,K‐ATPase protein abundance at the plasma membrane, and is therefore a critically necessary step in the cellular adaptation and survival in hypoxic conditions.Support or Funding InformationSupported in part by HL‐71643 and HL‐48129, AG‐49665

Molecular O2 and CO2 are the primary substrate and product of oxidative metabolism respectively. Hypoxia (low O2) and hypercapnia (high CO2) are co‐incidental features of the microenvironment in a diverse range of pathophysiologic states including cancer, respiratory disease and inflammation. Hypoxia inducible factor (HIF) is the master transcriptional regulator of the response to low oxygen. Despite the close relationship between O2 and CO2 levels, the potential impact of CO2 on HIF remains unclear. Recent studies have demonstrated the ability of CO2 to regulate gene expression. The purpose of this work was to investigate the effect of hypercapnia on the HIF pathway.HIF‐2α protein and the HIF targets erythropoietin and carbonic anhydrase IX were decreased by hypercapnia in vivo. Hypercapnia suppressed HIF‐1α and HIF‐2α protein stabilization in a variety of cell lines of diverse origin. High CO2 diminished the activity of HIF‐dependent genes. CO2 did not impact the canonical, oxygen‐dependent HIF degradation pathway. Hypercapnia caused intracellular acidosis, and levels of HIF‐1/2α protein were lower when intracellular pH (pHi) was reduced by means other than CO2. Low pHi is required for optimal lysosomal degradation and a lysosomal inhibitor antagonised the ability of hypercapnia to reduce HIF‐1/2α.In conclusion, we have demonstrated a novel CO2‐dependent suppression of HIF signalling. As HIF regulates the transcription of a host of genes that have angiogenic, glycolytic and inflammatory functions, manipulating HIF with CO2 could be exploited for therapeutic gain.Support or Funding InformationThis work was supported by a grant from Science Foundation Ireland (11/PI/1005)

Molecular oxygen and carbon dioxide are the primary gaseous substrate and product of oxidative metabolism, respectively. Hypoxia (low oxygen) and hypercapnia (high carbon dioxide) are co-incidental features of the tissue microenvironment in a range of pathophysiologic states, including acute and chronic respiratory diseases. The hypoxia-inducible factor (HIF) is the master regulator of the transcriptional response to hypoxia; however, little is known about the impact of hypercapnia on gene transcription. Because of the relationship between hypoxia and hypercapnia, we investigated the effect of hypercapnia on the HIF pathway. Hypercapnia suppressed HIF-α protein stability and HIF target gene expression both in mice and cultured cells in a manner that was at least in part independent of the canonical O2-dependent HIF degradation pathway. The suppressive effects of hypercapnia on HIF-α protein stability could be mimicked by reducing intracellular pH at a constant level of partial pressure of CO2. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase that blocks lysosomal degradation, prevented the hypercapnic suppression of HIF-α protein. Based on these results, we hypothesize that hypercapnia counter-regulates activation of the HIF pathway by reducing intracellular pH and promoting lysosomal degradation of HIF-α subunits. Therefore, hypercapnia may play a key role in the pathophysiology of diseases where HIF is implicated.

The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1(-/-) mice exposed to high CO2 did not develop muscle atrophy. AMP-activated kinase AMPK), a metabolic sensor, was activated in myotubes exposed to high CO2, and loss-of-function studies showed that the AMPK alpha 2 isoform is necessary for muscle-specific ring finger protein 1 MuRF1) up-regulation and myofiber size reduction. High CO2 induced AMPK alpha 2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPK alpha 2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.